miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences

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Sara dos Santos Teixeira da Silva

miRNA as biomarker for body-fluids identification

Its role in Forensic Sciences

Dissertação de Candidatura ao grau de Mestre em Medicina Legal submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto.

Orientador – Doutor Rui Medeiros Categoria – Professor associado Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.

Co-orientadora – Doutora Ana Luísa Teixeira

Afiliação – Grupo de Oncologia Molecular e Patologia Viral, Centro Investigação do Instituto Português de Oncologia do Porto.

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It is impossible to say how first the idea entered my brain; but once conceived, it haunted me day and night Edgar A. Poe

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Acknowledgment

No one should brave the underworld alone. Edgar A. Poe

So many persons got my back and give me such support throughout this extraordinary chapter in my life. I cannot afford to acknowledge all the surprising individuals that were there to help me or motivate me - there is why I do want to let you know that I am thankful for making this dissertation possible.

First, I would like to thank my advisor, Professor Rui Medeiros, for his orientation on this project and for the unconditional support he gave me - which greatly contributed to the evolution of this dissertation.

To my co-advisor, Dr. Ana Luisa Teixeira, always present to help me – without a shadow of a doubt, a role model to look up to.

I could not let to mention Professor Maria José Pinto da Costa for being such an example. Thank you for allowing me to do what I really love to do.

A special thank you to my Molecular Oncology group, especially to Francisca, for help me throughout this essay.

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To my Circle of Friends - Marta, Rosa, Telmo, Luís and Marcos – You guys are awesome! We are, without doubt, a hell of a team.

Vanessa, we started this journey together ten years ago and here we are … ending it (for now). You are nuts, you are silly, you are unbelievable and funny as hell - You are my CFF – Thank you!

To Cátia Lopes, Cátia Dias, Joana Vieira and Sissa Oliveira … I miss our time in class. We were pretty much crazy together.

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À ma tata, tonton et à mes deux GRANDES sœurs – Vous êtes tout simplement incroyables !

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Pour les deux personnes les plus importantes dans ma vie, à ma maman et à mon papa…

Pour tout ce que vous faites pour moi Pour tous ce que je suis grâce à vous

Pour tout ce que vous représenté pour moi …

Thank you Mercie

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9 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences

Abstract

In forensic investigation, body fluids represent an important support to professionals when detected, collected and correctly identified. Through many years, various approaches were used, namely serology-based methodologies however, their lack of sensitivity and specificity became difficult to set aside. In order to sidetrack the problem, miRNA profiling surged with a real potential to be used to identify evidences like urine, blood, menstrual blood, saliva, semen and vaginal secretions.

miRNAs are small RNA structures with 20-25nt whose proprieties makes them less prone to degradation processes when compared to mRNA which is extremely important once, in a crime scene, biological evidences might be exposed to several unfavourable environmental factors.

First of all, we proceed to an extensive gathering of information published till date and assessed a multitude of factors that have a potential aptitude to discrediting miRNA profiling, such as: methodological approaches, environmental factors, physiological conditions, gender, pathologies and samples storage.

Afterwards, we studied 4 miRNAs profiles within 2 body fluids – blood and urine – and settled whether or not those could be used as biomarkers for blood and urine identification.

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Resumo

Na investigação forense, os fluidos corporais representam um importante apoio para os profissionais quando detetados, coletados e corretamente identificados. Ao longo dos anos, várias metodologias foram abordadas nomeadamente tecnologias assentes em técnicas serológicas, no entanto, a sua falta de sensibilidade e especificidade tornou-se uma menos-valia difícil de contornar. De forma a dissipar o problema, o estudo dos perfis dos miRNAs surgiu com um verdadeiro potencial para identificar evidências tais como urina, sangue, sangue menstrual, saliva, sémen e secreções vaginais.

Os miRNAs são pequenas estruturas de RNA com 20-25nt cujas propriedades os torna menos propensos a processos de degradação quando comparado ao mRNA, o que é extremamente importante uma vez que, numa cena de crime, as evidências biológicas podem estar expostas a vários fatores ambientais desfavoráveis.

Em primeiro lugar, procedeu-se a uma extensa revisão bibliográfica publicada até a data e avaliou-se uma variedade de fatores com potencial para descreditar o uso de miRNA para a identificação de fluidos biológicos, tais como: uso de diferentes metodologias, fatores ambientais, condições fisiológicas, género, patologias e acondicionamento de amostras.

Em seguida, estudou-se o perfil de 4 miRNAs em 2 fluidos corporais - sangue e urina – com intuito de concluir se estes poderão ser ou não utilizados como biomarcadores para uma identificação fiável de sangue e urina.

Palavras-chave: Ciências Forenses, Fluidos Biológicos, Profiling, miRNAs, biomarcadores.

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1 - Introduction

1.1 - Analytical mind

It will be found, in fact, that the ingenious are always fanciful, and the truly imaginative never otherwise than analytic. Edgar A. Poe

Edgar Allan Poe, master of gruesome plots, released in 1841 the mysterious short-story "The Murders in the Rue Morgue". Poe created as his main character, Auguste Lupin whose, with no other than his mind and incomparable confidence, hunted clues using each one of his senses in order to solve unsolvable crimes. “The murders in the Rue Morgue” became the first detective fiction gender becoming a pillar to other works like Arthur Conan Doyle’s Sherlock Holmes.

Before human behaviour was recognized as an important part in criminal investigation, Poe narrated how the body language of a suspect, his accelerated heartbeat, rapid breathing and his guilt as a powerful tool to understand someone’s blame in a crime. The way he conducted his story, based in analytical thinking, “behavioural profiling” and intelligence, led the readers to participate alongside with the main character to solve crimes.

Edgar Allan Poe’s work projected forensic investigation to a wider public. With his books, Poe described the premises of scientific investigation through the sharp mind of his main character. Culminating, centuries later, in one of the most respected and fascinating science of all.

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1.2 - Forensic Sciences

Even in the grave, all is not lost. Edgar A. Poe The term “forensics” is a Latin word that means “belonging to the forum” - in ancient Rome, if a criminal case was brought up, it will be brought up before the “forum”. Constituted by witnesses and specialists, they will discuss the situation with great detail in order to determine guilt in civil or criminal disputes and persuade the forum of it [1].

Nowadays, forensic science could be defined as the application of a broad spectrum of scientific fields responsible for answer questions of interest in a legal system whether in civil or criminal actions [2]. Modern forensic science investigation, thrived by Locard’s principle - every contact leaves a trace - is capable to apply principles but also scientific techniques to analyse evidences recovered during a criminal investigation [3, 4]. It is also based on strict guidelines in order to ensure cautious and methodical collection, organization and analysis of information [5].

Forensic science can be dissected into several specialized divisions namely forensic pathology, anthropology, entomology, odontology, toxicology and genetics. Each with its own set of technologies, they contributes to solving crimes through investigative activities like determining the cause of death, finding missing persons, identifying suspects and profiling criminals [6-13].

1.3 - Evolution of Forensic Science

If you run out of ideas follow the road; you'll get there Edgar A. Poe

It is commonly though that forensic science is a relatively new science in our society. Far behind the glamorous ideology of the 21th_{century TV series,} criminal investigation is definitely not such a recent science as it may appear. The beginning of Forensic sciences, what we could call - pre-modern forensic science

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23 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences – was not exactly forensic science. Before the 17th_{century, for the most part of it,} it was not “real” forensic science, however, some of its background did develop on that epoch. By then, for crimes as murder or rape, there would be the confrontation of the accuser and whoever is being accused of that crime. The suspect would be tortured until its confession - which will ensure its guilt. On the other hand, if the suspect was able to resist the pain and have the strength to go through the torture it would be considered as innocent [14].

A pre-historic drowning of a hand with ridge patterns found in Nova Scotia and imprinted fingerprint in clay tablets used for business transactions in ancient Babylon, seem to point the origin of dactiloscopy for common use at 1000-2000 Before Christ [15-17]. Around year 250BC, a Greek physician, Erasistratus, observe that his patient pulse rates increased when he lied. His observations were considered as what would be the first lie detection test [18].

In 44 BC, Antistius, the personal physician of Julius Ceasar, realized the first recorded autopsy known. He concluded that even though Ceasar was stabbed 23 times, the second blow of the knife – the one that hit his heart – was the one that killed him [19, 20].

On the 3rd_{century, in China, Yi Yu Ji was published and has some} semblance of a science applied to criminal cases. One of the cases regarded in that publication was of a man that burned to death. His wife claimed that it was an accident, however, officials found that there was no ashes in his mouth which raised their suspicion. In order to elucidate their doubt, their burned 2 pigs, one alive and another already dead. The pig that was alive when burned has ashes in his mouth; on the other hand, the pig that was dead did not. Upon this fact, it was proven the culpability of the victim’s wife [21].

In 1248, still in China, was published the first forensic science book called as Hsi Duan Yu (the washing away the wrongs). This book discussed the differences between crimes as strangulation versus drowning and became an official manuscript for physicians [22-24]. Still that year, a man was stabbed to death with a sickle. Sung Tz’u, a medical examiner, gathered everyone in the village and had them laid their knives and sickle on the ground and waited. Over time, flies came and landed in a particular sickle distinguishing, among others, the murder weapon and by association, the perpetuator of the crime [25, 26].

The German Constitutio Bambergensis Criminalis, appeared in 1507, that highlighted the importance of physicians in cases of infanticides [27]. Also known

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24 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences as the Carolina, it was recognizes as the first criminal law body and punished actions as murder, robbery, manslaughter among others [28].

The Father of surgery, Ambroise Paré, wrote and published in 1575 Reports in Court. The French surgeon, already known for his studies on death wounds (pre and post mortem), infanticides, hanging, among others, launch through his manuscript a new era – The era of modern forensic pathology [29, 30].

Hydriotaphia, Urn Burial, wrote by Sir Thomas Browne in 1658 make for the first time reference to adipocere [31]. The English physician, also known as a pioneering forensic archaeologist, described the unknown process as a soap-like substance related with moist places. Although, the term adipocere will only surge in 1789 by Antoine François Fourcroy [32].

In 1687, Marcello Malpighi noticed the ridges, spirals and loops on fingertips, which would become fundamental for human identification [15, 33, 34]. However he did not acknowledge their function or importance as a mean of human identification [34].

In 1775, the Swedish chemist Carl Wilhem Scheele created a test able to detect arsenic poison in corpses [35, 36]. His work would be improved by Valentin Rose, who will discover a far more precise method to detect small amounts of arsenic [37].

Three years later, the first recognized documented use of physical matching – bullet wound to a suspect - was released in UK. John Tom was convicted for the murder of Edward Culshaw after a wad of paper found in the victim wound was complementary with the missing corner of the sheet in the possession of the suspect [38].

In Paris, the former convict Eugène François Vidocq created the first detective force in 1810 [39, 40]. The so-called Sureté of Paris was staffed with criminals and was specialized in criminal investigation [41]. His work established Vidocq as the father of modern criminology [41]. The Vidocq Society, created in honor of Vidocq is composed by forensic professionals and private citizens who directed their work to solve cold cases [42].

1813 unveiled one of the most important individual in forensic toxicology – Mathieu Orfila [43]. Considered as the father of modern toxicology, Orfila published Traité des poisons which was the first report on poison detection and started to be used as a guideline for murder cases [44].

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25 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences In 1828, William Nicol invented the polarized light microscope that led Henri-Louis Bayard in 1839 to formulate the first procedures for microscopic detection of sperm [45-47]. Later that year, James Marsh, a Scottish Chemist, was the first person to testify using toxicological evidences in a criminal trial [22, 48, 49]. That is when we start to see the merging between science and law - which is the principle of forensic science.

1850s to 1860s settled for a development of photography which improved records in forensic science more specifically in prison system [50].

In 1879, the French anthropologist Alphonse Bertillon introduced the Bertillon system which is also known as anthropometry [51]. He used a large amount of measurements of the body to identify people by their physical appearance [52, 53]. However, this worked relatively well until a particular case – the Will West case – put it to an end (figure 1). In 1903, a man called Will West was put in prison. The person who was processing him asked if Will West had already spent time in prison before which he answered negatively. The clerk went to the record and founds out that there was another person in that prison by the name of William West that looked nearly identical. When put in the Bertillon system, their measurements were also practically identical. This coincidence put an end to the commonly used method of identification [54-56].

Hans Gross, in 1893, published Criminal Investigation that discussed the benefits of botany, chemistry, fingerprinting, geology, microscopy, physics and zoology in criminal investigations [57, 58].

The Nobel Prize winner Karl Landsteiner discovered in 1901 human blood groups which would be adapted later on by Max Richter for blood stains typing [59-62].

Figure 1 – The West Case - Photographs of both Will and William West displaying their

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26 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences The American President, Theodore Roosevelt, established in 1905 the Federal Bureau of Investigation (FBI) whereas FBI director J. Edgar Hoover opens the FBI laboratory in 1934 [63, 64].

In 1910, Albert Osborn published Questioned Documents which is still a reference when determining document forgeries [65, 66].

Another important personality in the forensic field is Edmond Locard. Between 1877 and 1966 the French doctor and criminologist opened the very first crime laboratory in France but most importantly, he created the Locard’s Exchange principle. Basically, this principle relies on the fact that anytime 2 things make contact, there is an exchange of material [67, 68]. There was a case involving possible forged coins that had 3 suspects. Locard asked to see the suspect’s clothes that they were wearing when arrested and was able to find small metal fragments on them. Locard match those metal fragments to the forged coins resulting in the condemnation of the suspects [69].

The American chemist Walter McCrone, between 1916-2002, was the leading expert in microscopy and was the one asked to examined the famous Shroud of Turin and the Vinland map [70-72].

In 1990, the FBI created its personal database of genetic profiles from convicted criminals and those associated with unsolved crimes. Named Combined DNA Index System – CODIS – the program constitute a real step forward in forensic genetic field (figure 2) [48, 73].

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1.4 - Forensic Genetics

Invisible things are the only realities Edgar A. Poe

1.4.1 - Study Case

In 1987, days after Thanksgiving, the detective Joe Horgas was on duty when he received a call for a homicide case in Arlington suburb, outside Washington D.C. He and the other officers proceeded through the house and found the dead body of the 44 years old Susan Tucker laid face down on the bed of the master bedroom. Naked with just a blue sleeping bag next to her, she had her hands tied behind her back with a brand-new rope going from her hands up to her neck. By the time of the murder, Susan’s husband was in Great Britain visiting his family in Wales, where the couple has been planning to move. Agent John Coale and Rick Schoembs from Arlington County police department investigate the crime scene and documented it with close-up photography. On the floor nearby, an empty purse with its content scattered on the floor. The officers zeroed-in what they thought to be the point of entry of the assailant. Outside of the house there was a patio and just beneath it a narrow basement window that was found broken. The officers collected all the broken glass they could find. It is important to acknowledge that there is always a transfer of material between 2 objects – Locard’s Exchange principle. If the assailant did entry the house by that window, the glass would present some kind of evidence occasioned by an exchange of assailant-glass material.

Curiously, a similar murder occurred 3 years earlier. Carolyn Hamm was raped and murdered only 3 blocks away from Susan Tucker’s house. Just as Susan Tucker case, the assailer entered her house by a narrow opening and the content of her purse was scattered on the floor yet nothing of value was missing. Because of it, Joe Horgas pulled the Carolyn Hamm file in order to get leads to his case. Carolyn Hamm’s file was closed, a man was convicted for her murder and already behind bars but doubts still remained. Everyone who worked in the case didn’t believe that the suspect – David Vasquez – was responsible for the crime or at least, it was not the only responsible. David Vasquez was serving 34 years of reclusion, he plead guilty in order to avoid death penalty. Then, 3 years later, the same exact crime happened. Detective Joe Horgas believed that the perpetuator of Carolyn Hamm’s murder was the same of Susan Tucker’s. Despite the

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29 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences similarities, Horgas had no leads and any fingerprints were found. However, Horgas had hopes for the other evidences. Once in the laboratory, the blue sleeping bag, Susan’s nightgown and a washcloth with smears on it were analysed and it was found 4 different stains just in the nightgown. They tested them with acid phosphatase and the purple colour indicated the presence of semen but to confirm it, a small portion of the stain was examined microscopically. After positively identified, remained the questions: to whom it belongs? In 1987 the only way to connect semen to a rapist was, first of all, by determining if any blood was present in it. Is so, the blood could be typed. In that case, the blood type was O and matched neither the victim nor her husband. The results narrow down the field of possible suspects, however, the stained washcloth held an additional piece of the puzzle – 2 pubic hairs. The pigments granules analysis enabled the determination of the race of the suspect and in that case, the pubic hair came from a black man.

At the same time, in Richmond (2 hours from Harlington), a serial murderer known as the South Side strangler was on the loose. Responsible for the rape and murder of 3 women within the preceding 3 month, could the South Side strangler be responsible for the murders in Arlington? Detective Horgas went to Richmond to compare notes and the similarities were impressive. All 3 victims were tied-up and strangled but most importantly, in all cases, the assailant entered by the window. Later, lab tests confirmed that the semen stains found in the 3 murders were identical in blood type to those found in Susan Tucker’s nightgown. However, a common blood type is hardly enough to implicate a killer.

In 1987, Deoxyribonucleic acid (DNA) testing was not widely used in forensic labs. Detective Horgas sent the semen stains found in the nightgown of Susan Tucker to a laboratory in New York. Richmond police also submitted semen samples from the 3 murders. Back then, the process had a limited sensitivity, it was required a large amount of biologic sample and its analytic study could take to 6-8 weeks.

Due to the gap of 3 years between the murders, detective Horgas concluded that the suspect had probably been in prison and searched for someone who got arrested after Carolyn’s murder and got released sometime in 1987. Taking this into account, Timothy Spencer became a suspect. Released of prison in 1987, he went to a halfway house in Richmond 2 weeks before the first crime.

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30 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences On January 20th_{1988, Timothy Spencer was arrested. At the time they had} little evidence - just the remains of glass from the broken window. After his arrest the police went to his room in the halfway house where they found his pants, ski mask and an army jacket. Lab tests were conducted in a small piece of glass found in the suspect jacket and compared to the shards of the broken window of Susan Tucker’s house. Even if both had the same optical proprieties, this evidence was not enough to convict the suspect because the glass was not unique enough to distinguish it from hundreds of other windows. The only physical evidence that could link Timothy Spencer to the crime was DNA.

After his arrest, a sample of his blood was taken and his DNA profile was drawn. Four months after the murder of Susan Tucker, the results were completed – the DNA from the crime scene matched with the Timothy Spencer’s DNA. In July 1988, Timothy Spencer trial begun and condemned him to death penalty. In 1989, David Vasquez received a pardon and was released.

This case was the first to convict a suspect of a capital murder in the United States based on DNA profiling [74-76].

1.4.2 - Genetic Background

Every living organism has their own individual DNA and its singularity allows us to distinguish humans from animals or even plants [77]. Most importantly, due to the different combinations of DNA sequences, the study of each “genetic prints” allows an individual identification among humans [77, 78].

DNA can be found in humans cells, namely in blood, hair and sperm – that are also the most commonly samples found in crime scenes [5]. After correctly collected and analysed, it is possible to determine a DNA profile allowing individual identifications [78].

In order to create a genetic profile, a certain amount of genetic material is required. The genetic material can be easily found however, adverse conditions as heat, pH, drought, among others can present negative effects and deteriorate the samples preventing its analysis and posterior DNA profiling [79]. In a crime scenario, after evidences collection, it is necessary to gather samples from the suspects in order to compare both genetic profiles and conclude whether or not the suspects are linked to the crime. Nevertheless, the genetic profile needs to be compared with the victim DNA in order to remove the possibility that the

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31 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences “suspect” DNA was the one provided by the victim. After processed, the information needs to be interpreted. If both evidence and suspect sample profiling matches, it is required to determine the probability that the profiles correspond with another profile in random population [80].

This way, the use of DNA profiling in criminal investigation can bring benefits to the society by helping to solve crimes, exonerate innocents and assisting in the enforcement of the rule of law.

1.5 - Importance of body fluids identification

Believe only half of what you see and nothing that you hear. Edgar A. Poe

Serological test allows the detection and identification of body fluids in both native form or as a residue left at a crime scene [15]. Items on which body fluids are thought to be present are submitted to laboratories for serological test and DNA analysis [81]. Before any analysis, it is needed to choose whether the items go through serological test or if they are sent directly for DNA analysis [82]. Indeed, each body fluid requires a different molecular methodology in order to get a more reliable product to conduct a DNA analysis. For example, DNA extraction processes are different for blood and urine. If we conducted the protocol of blood extraction in urine samples it may result in a reduced quality of the isolated DNA, which can compromise the DNA analysis [77, 82].

If fresh blood is fairly easy to identify, bloodstains on grass changes colour quickly, making difficult its identification [82]. Though, this process happens with other biological evidence such as seminal fluid, urine, saliva and so on, when dried or washed. It is why all evidence goes through serological screening first in order to reliably identify the item and ultimately proceed to the molecular process to obtain the most high-quality sample possible. However, samples with trace amounts of DNA goes straight to DNA analysis [79, 82].

Whereas the majority of cases processed involved violent crimes there is been an increase of cases involving DNA to assist in solving property crimes.

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32 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences Biological evidences are usually left by the burglar. For example, a burglar may injure himself and leave blood at the crime scene or, as an act of vandalism, can relieve himself on the items of the house [48, 83].

As a matter of fact, a complainant’s body fluid present on items belonging to a suspect - or vice-versa - holds the most probative value [15]. For example, in cases of sexual assaults, the detection and identification of semen from a sexual assault kit is crucial to support a claim of the same purpose.

In order to be sure, the identification of body fluids through serological analysis is accomplished through presumptive and confirmatory tests. Presumptive tests rely on methodologies that are sensitive and performed quickly, yet they are not specific to the body fluid. Those tests can only indicate if the fluids might be present and do not unequivocally states its presence [77]. On the other hand, confirmatory tests are indeed specific to the body fluid we seek to identify. As presumptive tests, confirmatory testing is sensitive however, it takes a lot more time [77].

1.5.1 - Blood Identification

A comatose teenage female was discovered to be pregnant and the suspected father was no other than a juvenile friend of the victim’s brother. In order to elucidate whose the father was, the aborted fetus was submitted along with the victim’s and suspect’s blood for a paternity test. Blood samples from the victim’s brother and father were also submitted. To the general surprise, the suspect was

excluded as the father of the fetus, however the results showed that the brother was the one who fathered the fetus.

Adapted from Fisher et al. [84].

The identification of blood can be key-evidence in a wide range of possible situations. Blood identification is essential to many homicide investigations but also in cases involving aggravated assaults, burglary and sexual assaults [85]. The presence of blood can support the complainant’s or suspect’s version of alleged events establishing culpability or innocence during a criminal proceeding. Before an extensive blood loss, its detection may not be difficult. However, in some cases where only a very small amount of blood is transferred, its

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33 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences detection may be very complicated. In cases where the crime scene is been cleaned-up and no blood is visible to the naked eye it is important to rely on serological tests that could allow us to detect and reliably identify blood from clothing, floors, grass or any other surface.

The bright red distinctive colour of blood is derived from haemoglobin. Yet, when it dries, the colour darkens to red-brown and finally to brown, which can difficult its detection and identification.

When an unknown stain is found at a crime scene, the first process we need to use is the so-called presumptive tests. Those tests usually help to elucidate some important questions as: is it blood? or Is it human blood? [86]. Idealistically, presumptive test must always be supported with confirmatory testing (figure 3).

Till date, there are 3 main presumptive tests for blood: Phenolphthalein, Tetramethylbenzidine and Luminol test [15].

Phenolphthalein test, also known as Kastel Meyer test, is the most frequently used presumptive test for blood [87, 88]. When the chemicals are added, they will react with the haemoglobin present in blood causing the formation of a bright pink colour within 10 to 20 seconds [89]. As its main advantage, this particular technique is very sensitive and highlights stains that are barely visible or even imperceptible to the naked eye [87, 90]. As the others presumptive tests, this particular one does have a major disadvantage – it can easily create false positives once, substance like rust, cooper, among others can also react resulting in a bright pink colour [87].

Tetramethylbenzidine test works the same way as Kastel Meyer, but in this particular case it turns into a blue-green colour instead of a bright pink dye [87, 91]. When compared, tetramethylbenzidine test present a greater level of specificity yet, it does not work as well in diluted blood stains, which is a substantial and important limitation[91].

When blood is suspected to be present but invisible to the naked eye, it would not be reasonable to test the whole item on which the stain may be present. In such occasions, luminol testing is used to indicate nonvisible blood stains [15]. Instead of producing colour change reactions, this chemical presumptive test leads stained areas to emit light [86]. This test is particularly sensitive – even more that the 2 precedent tests previously referred [15]. One disadvantage attached to this test is the same verified with the phenolphthalein test, the occurrence of false positives [67]. In addition to those already named

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34 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences previously, it also react with bleach and others cleaning fluids which is a problem once it may interfere and create false results on surfaces that have been cleaned [92]. Moreover, when we spray a stain with the chemical, it dilutes the stain. In cases where the stain is already weak, its further dilution may lessen the chances of obtaining a good DNA profile which is another significant drawback to that test.

Nowadays, the Rapid Stain Detection of human blood (RSID™-blood) and the ABAcard® are the most commonly used confirmatory tests in forensic laboratories [87]. Rapid Stain Detection kit enables blood identification through the detection of human glycophorin A, which is present in erythrocytes membrane [73]. Likewise, ABAcard® kit it is very similar to the precedent but detects the presence of human haemoglobin instead of glycophorin A [73, 85].

1.5.2 - Semen Identification

Before the complaint of a sexual assault to a child, a DNA profile was recovered from samples taken from a child’s underwear and bedding. His step-father, considered as suspect in this case, argued that the source of DNA was recovered from his skin cells deposited from a casual and frequent contact with the clothes and beddings of the child. For instance, if serological tests were done, in order to elucidate from which tissue the DNA was recovered, and concluded that his DNA was originated from semen and not from skin cells, those results would definitely

plays a key-role to support the allegation of sexual assault.

Semen is an extremely important body fluid in cases of sexual assaults [91]. As for example, the identification of semen on vaginal smears swabs or on a victim’s clothing may be of value in corroborating the victim’s claims.

Figure 3 - Presumptive and confirmatory tests for blood. Representation of: A- Phenolphthalein test. B-

Tetramethylbenzidine test. C- Luminol testing; D- Device used for both ABAcard® and RSID™-blood confirmatory tests.

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35 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences It is important to underline that not all men produce spermatozoa. Some who have birth defects, have had a vasectomy or as the result of a disease either do not contain or contain very few spermatozoa in their seminal fluid [87]. For that reason, in forensics, 2 major components of semen are particularly important for its identification: seminal fluid and spermatozoa [87].

The most frequently used presumptive test for the detection and identification of seminal fluid relies on the detection of the enzyme acid phosphatase (figure 4) [93]. This enzyme, present in seminal fluid, is also present in others body fluids such as urine, saliva or vaginal secretion yet, in lowest concentrations [87]. The internal concentration of the enzyme allows it be used as a presumptive test for seminal fluid identification, however, its presence in other body fluids leads to a tricky complication: false positives. Acid phosphatase is identified using the brentamine spot test which alters the colour of the tested area into a bright purple colour in a positive case [15, 91]. The absence of colour change not only may indicate that no seminal fluid is present but also may indicate that the level of the enzyme is lower than the detection limit of the test. This is the reason why, when this methodology is used, it is indeed necessary to use additional tests to confirm the presence or absence of seminal fluid [15].

It wouldn’t be reasonable to test a large item with the acid phosphatase test to detect nonvisible stains. In order to visualise those stains, an alternate light source is used to pre-screen the item and detect areas that could be processed for acid phosphatase detection (figure 4) [87]. However, not only semen does fluoresce when exited with alternate light but other like saliva or urine does too - hindering its reliability [15].

Items that have been tested presumptively positive can be confirmed by 2 different processes: either by a chemical detection of a specific protein of semen or through a microscopic detection of spermatozoa (figure 4) [48, 87].

Identify spermatozoa microscopically is an undeniable proof that semen was present in the analysed item. Positively tested swabs can be smeared onto a microscope slide and stained by what is commonly called the Christmas tree stain [87]. Composed by two different dyes – nuclear fast red (red colour) and picroindigocarmine (green colour) it enables the visualization of spermatozoa. The head of the spermatozoa will turn red while the tail, if present, will turn green - hence the name of Christmas tree [48, 87]. Nonetheless, seminal fluid belonging to a vasectomise individual does not have spermatozoa therefore, in this situation, microscopic methods do not allows the identification of seminal

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36 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences fluid [15]. Moreover, the time necessary to microscopically analyse the items is considerable - representing an undeniable drawback when compared to the protein confirmation method.

The protein confirmation method can be done through the Rapid Stain Detection (RSID™-semen). Rapid Stain Detection kit enables semen identification through the detection of prostate-specific antigens (PSA) and when tested positive, serves as an undeniable confirmatory test for semen [87].

1.5.3 - Saliva Identification

An executive in a major company received threat letters and suspected one of his employees who also had an affair with his wife. Though, no arrest or search warrant was up due to the lack of evidence. Later, the chief of the police received

a mailed bomb coupled with a threat letter. The saliva present on both treat letters were subjected to a DNA typing and finally compared to the saliva residue

found in love letters that the suspect sent to the executive’s wife. The DNA types matched and were enough for a search warrant for a sample of blood of the suspect. After confirmation, the suspect was charged with attempted of murder.

Adapted from Fisher et al.[84].

Secreted by the parotid, sublingual, submandibular salivary glands and the mucous glands of the oral cavity, saliva is responsible to keep the mucous membrane moist. It is also responsible for salivary digestion of carbohydrates once the saliva principal component, α-amylase, convert starch into maltose [94].

The transfer of such material can be resulting of direct contact as in food when eating, cigarette butts, straws, drink vessels, envelopes, stamps, bite marks or in cases of oral sex assaults. On the other hand, saliva gathered from an object

B C D

A

Figure 4 - Presumptive and confirmatory tests for semen. Representation of: A- alternate light source test; B- Acid

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37 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences as a telephone – mouth piece - result of projected biological material, is a good example of indirect transfer [77].

Currently, saliva screening is mainly done through presumptive tests (figure 6) [95]. Alternate light can be used as a tool for saliva identification, usually in cases where biological fluid are searched in clothing [96]. When excited with alternate light, saliva fluorescence highlights its presence among other biologic samples [96, 97]. However, there is a pretty obvious limitation to that test. Just like saliva, seminal fluid also responds to alternate light resulting in fluorescence (figure 5) [87]. To that point, if the laboratory analyst knows where to look, the problem dimension can be reduced - increasing the probability that the fluorescent stain is connected with saliva. Nonetheless, if it goes the other way and the analyst does not know what he is looking for, he will face a major problem – he will not be able to identify any saliva stains. Due to the fact that many other fluids or even substances can fluorescent with alternate light, this presumptive test is used only to identify areas for additional examinations through other screening tests.

Another set of presumptive test for saliva and currently the most widely used is based on amylase detection which is an enzyme that is found at high levels in saliva. The Phadebas® test is used in forensics to identify stains containing amylase and it is performed using paper impregnated with a reagent that dissolves into the paper releasing a blue dye when contacting with amylase [98, 99]. As any other presumptive test, there is some serious limitation to the methodology – Phadebas® test does not differentiate human saliva from other species and, as it was referred before, amylase can be found in other fluids other than saliva and thus this test can identify stains that are not saliva. In order to sidetrack the problem related with species, another presumptive test called Rapid Stain Detection for saliva (RSID™-saliva) was released [100]. RSID™ test, unlike Phadebas®, only recognize human α-amylase [101].

Figure 5- Stains detected by alternate light source – this presumptive test allows the detection of saliva however, other body-fluids do also shows.

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38 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences To our knowledge, there is no confirmatory test for saliva identification. However, some laboratories choose to run all 3 tests in order to increase the probability to reliably identify human saliva.

1.5.4 - Urine Identification

Two young brothers accused their neighbour of child molestation, claiming that he gave them pills in order to make them dozy. Blood and urine of both kids

were collected for a toxicological screening. Before negative results, the children confessed that they made-up the entire story because they did not like their

neighbour.

Adapted from Fisher et al. [84].

The urinating act is commonly found as an act of vandalism and, when gathered, urine can be a source of trace DNA [48]. However, this body fluid is found to be particularly good for drug screening, especially drugs of abuse once nearly all drugs are eliminated in it [48, 102].

There are several simple and fast presumptive tests for urine. The oldest test is undeniably related with its characteristic colour and odour [82]. A simple heating of the samples can emphasise the odour and help with the identification process [82].

The easiest presumptive test is a chemical test named Jaffe colour test. This test is based on the detection of creatinine through the formation of creatinine picrate. A drop of water extracted from the putative stain is mixed with a drop of saturated picric acid followed by a drop of 5% NaOH. The apparition of a bright orange colour indicates the presence of creatinine [82, 103].

Another presumptive test relies on the detection of the major component of dried urine - urea. In the presence of the enzyme urease, urea is cleaved and

A B C

Figure 6 - Presumptive and confirmatory tests for saliva. Representation of: A- alternate light

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39 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences formed ammonia which can be easily detected by bromothymol blue. This mapping process uses large sheets of dampened filter paper that allows urine stains to be located when the paper turns bright blue [104]. Due to the limitations associated with those presumptive tests, it is essential to develop more sensitive and specific methodologies.

1.6 - Genetic approach – a broad confirmatory test?

Experience has shown, and a true philosophy will always show, that a vast, perhaps the larger, portion of truth arises from the seemingly irrelevant. Edgar A. Poe

As previously stated, from a forensic point of view, body-fluids identification are crucial for evidential value but also to guarantee an accurate handling of samples, culminating in a reliable DNA profiling. As it is evidenced, there are a lot of presumptive tests available however, confirmatory tests are way scarcer or simply inexistent. If the reliability of results depends primarily from a correct identification of a certain body fluid, can we really relies ourselves on presumptive tests?

Fact is, for some body fluids like menstrual blood, there is no confirmatory test drawn-up till date. Forensically speaking, there is no shadow of a doubt that menstrual blood is important. If we focus on a sexual crime complain, the presence of blood or menstrual blood in the victim’s underwear do not have the same impact. Quite the contrary, a correct identification of the stain could be considered as a major evidence to blame or exonerate the suspect.

Currently, we stand in a molecular epoch where everything seems to find its explication and resolve through a DNA approach. Following this line of thought, it would be reasonable to believe that the ultimate confirmatory test would be based in our genetic background.

If DNA profiling allows the identification of individuals through their respective DNA signatures, it does not identify the type and source of the evidence. Its lack of distinctive signature for each type of body tissue withdraw DNA profiling as a possible candidate as a confirmatory test.

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40 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences Looking closely at the central dogma of molecular biology, DNA is transcribed into RNA which is translated in protein. Interestingly, RNA does have a specific pattern related to the tissue source. Facing that, is it reasonable to believe that RNA could be a key-factor to answer the dilemma?

1.6.1 - Potential of RNA

Different cell types present in body fluids have different functions and thus, they require a different cluster of functional proteins [105, 106]. Once messenger ribonucleic acid (mRNA) is responsible for the translation of DNA genetic code into proteins, it is logical to consider that different cell types have also a different mRNA pattern expression.

Advantages linked to mRNA profiling are undeniable. It has a great specificity, for example, for any tissue we choose, we are able to find mRNAs that are tissue specific [107]. It is also possible to simultaneously analyse a multitude of markers through common assays. Decreased sample consumption, co-extraction of both DNA and RNA and the automation of the laboratorial procedures are a serious improvement that enhances this methodology [108].

Some important results were published from precedent reports as it is shown on table 1.

Table 1 – overview of some mRNA published as biomarkers for body fluids.

mRNA REFERENCES

BLOOD beta-spectrin (sptb) [109]

porphobilinogen deaminase (pbgd) [109] aminolevulinate synthase (alas2) [110]

SALIVA statherin (stath) [107, 109, 110]

histatin 3 (htn3) [107, 109, 110]

mucin 7 (muc7) [110]

SEMEN protamine 1 (prm1) [109, 110]

protamine 2 (prm2) [107, 109, 110] VAGINAL

SECRETIONS human beta-defensin 1 (hbd-1) _{mucin 4 (muc4)} [109, 110] _{[107, 109, 110]}

keratin 16 (krt16) [107]

MENSTRUAL

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Despite the accomplishment of mRNA profiling, mRNA susceptibility to degradation by physical or chemical factors has always been problematic [111].

The use of RNA presents itself with great potential. Theoretically, mRNA seems to be reliable as a confirmatory test however, its susceptibility is a major impediment. That is why forensic researchers decided to look towards the most

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2 - Aims

The aim of the present work was to study miRNA profiles that could be used for the positive identification of some body fluids commonly encountered in criminal investigations.

The first aim consists to a systematic review of all information available till date about the use of miRNAs as a possible biomarker for body-fluids identification. We also acknowledge possible variables that could undermine its potential as a confirmatory test.

The second aim was to determine the relative expression of miRNAs in 2 forensically important body fluid – blood and urine - and extent the study to acknowledge the importance of the quality of the sample and its reflection in miRNA profiling.

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3

Material

and

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3 - Material and methods

Chapter 1 – Systematic Review:

“Forensic miRNA: Potential biomarker for body fluids?”

In forensic investigation, body fluids represent an important support to professionals when detected, collected and correctly identified. Through many years, various approaches were used, namely serology-based methodologies however, their lack of sensitivity and specificity became difficult to set aside. In order to sidetrack the problem, miRNA profiling surged with a real potential to be used to identify evidences like urine, blood, menstrual blood, saliva, semen and vaginal secretions. MiRNAs are small RNA structures with 20-25nt in length that make them less prone to degradation processes when compared to mRNA which is extremely important once, in a crime scene, biological evidences might be exposed to several unfavorable environmental factors. Recently, published studies were able to identify some specific miRNA, however their results were not always reproducible by others which can possibly be the reflection of different workflow strategies for their profiling studies. Given the current blast of interest in miRNAs, it is important to acknowledge potential limitations of miRNA profiling, yet, the lack of such studies are evident. This review pretends to gather all the information to date and assessed a multitude of factors that have a potential aptitude to discrediting miRNA profiling, such as: methodological approaches, environmental factors, physiological conditions, gender, pathologies and samples storage. It can be asserted that much has yet to be made, but we pretend to highlight a potential answer for the ultimate question: Can miRNA profiling be used as the forensic biomarker for body fluids identification?

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Chapter 2 – miRNA profiling:

“Circulating miRNA signatures for blood and urine identification”.

In this report, we choose to test the presence and expression levels of four miRNAs in both blood and urine samples obtained from 50 healthy individuals from Portugal and study their behavior within those body fluids

.

We conducted a miRNA expression profiling study of 50 healthy adult individuals (34% males and 66% females). Our group of study was composed by Caucasian individuals with a mean age of 42, 7 years old, with no major pathological condition. Peripheral venous blood and urine were collected from each subjects following the obtainment of a written informed consent from all subjects. After samples collection, the samples were processed, miRNA extracted and a cDNA library was created.

Afterwards, we proceed to get a relative quantification of our four miRNAs selected - miR-127, miR-221, miR-222 and RNU48 - through real-time PCR.

Statistical analysis was carried out by the computer software IBM®SPSS®Statistics (Version 22.0). In order to assess any statistical alterations in our normalized miRNAs expression essay we used 2−ΔΔCt_{method and Student's t test.}

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4 - Chapter 1

Abstract

In forensic investigation, body fluids represent an important support to professionals when detected, collected and correctly identified. Through many years, various approaches were used, namely serology-based methodologies however, their lack of sensitivity and specificity became difficult to set aside. In order to sidetrack the problem, miRNA profiling surged with a real potential to be used to identify evidences like urine, blood, menstrual blood, saliva, semen and vaginal secretions. MiRNAs are small RNA structures with 20-25nt whose proprieties makes them less prone to degradation processes when compared to mRNA which is extremely important once, in a crime scene, biological evidences might be exposed to several unfavorable environmental factors. Recently, published studies were able to identify some specific miRNAs, however their results were not always reproducible by others which can possibly be the reflection of different workflow strategies for their profiling studies. Given the current blast of interest in miRNAs, it is important to acknowledge potential limitations of miRNA profiling, yet, the lack of such studies are evident. This review pretends to gather all the information to date and assessed a multitude of factors that have a potential aptitude to discrediting miRNA profiling, such as: methodological approaches, environmental factors, physiological conditions, gender, pathologies and samples storage. It can be asserted that much has yet to be made, but we pretend to highlight a potential answer for the ultimate

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54 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences question: Can miRNA profiling be used as the forensic biomarker for body fluids identification?

Keywords: Forensic science, Forensic serology, body fluids, miRNA profiling, biological biomarkers.

1- Introduction

MiRNAs are small non-coding RNAs with approximately 22 nucleotides of length that seems to regulate a major part of human genes when combined with the RNA-induced silencing complex

[

1-4]. When this happens, miRNAs controls gene regulation by degradation of the mRNA through cleavage or by preventing protein synthesis [

5]

. Well conserved in eukaryotic organisms, they are involved in several cellular processes such as apoptosis, development, differentiation and proliferation

[

6-10]. In recent years, numerous studies showed that miRNAs profiling had a significant role as disease biomarkers, as a powerful tool to understand gene regulation mechanisms such as development mechanisms and gene regulatory networks; and also, due to its tissue specific pattern, in forensic sciences allowing the identification of body fluids [

11, 12]

.

Ambros and co-workers identified the first miRNA, miR-lin-4, in 1993

[13]

. They observed that this miRNA was responsible for the timing of development events of C. elegans larvar stages, they also discovered that lin-4 was not able to code a protein, but instead encodes what they called small RNAs

[13]

. Later, a second miRNA, let-7, was described by Pasquinelli et al. and was confirmed its presence in human tissue among different species. This observation demonstrated that these miRNAs were conserved through lineages, making that way, and for the first time, an allusion of an extended phenomenon

[14]

.

If DNA profiling allows the identification of individuals through their respective DNA signatures, it does not identify the type and source of the evidence. Based on the theory that each type of body tissue has a distinctive RNA signature, mRNA profiling surge as an advantageous procedure to identify relevant human body fluids. Despite the achievement of mRNA profiling, the mRNA susceptibility to degradation by physical or chemical factors has always been problematic

[15]

. In 2009, miRNA profiling to forensic field was introduced by Hanson et al., revealing the ability of miRNAs to identify different body fluids

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55 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences through miRNA signatures

[16]

. For example, when a certain miRNA is specific to a human tissue and cannot be found in another sample or when its concentration is significantly elevated and low in other samples, those characteristic profiles of miRNAs may allow the identification of human body fluids such as blood, menstrual blood, semen, saliva and vaginal secretions from others human tissues

[16-21]

. Argonaute proteins are catalytic component of the RISC complex, responsible for the biological process called RNA interfering [

1, 2, 4]

. Their tight relationship with miRNA makes them much more stable to degradation processes when compared with mRNA culminating in a superior discriminatory potential, especially in challenging conditions

[22, 23]

.

Through this review we pretended to gather all information available till date about the use of miRNA as a possible biomarker for body-fluids identification. But also, acknowledge possible variables that could undermine its potential as a biomarker.

2- miRNA as potential body fluid biomarkers

Body fluids constitute a beneficial assistance for forensic pathologists and researchers to present means of identifying the perpetrator of a crime and describe how an individual died or suffered through an assault. After being detected and recovered, biological traces need to be identified but some biological stains are difficult to undoubtedly identify, for example, venous blood versus menstrual blood. Through the years, many types of approaches such as chemical tests, immunological tests, microscopy and spectroscopic methods have been used to identify body fluids, however, some of them, like luminol for blood, are presumptive

[24]

. Henceforth, the potential of miRNA as a biomarker is being studied by means of a molecular genetics-based approach but can miRNA be considered a good biomarker for biological fluids? Idealistically, biomarkers should be able to fulfill a quite imperative number of characteristics

[25]

. It needs to be available to analyze through non-invasive methods; have a long half-life in samples; be unalterable by physical or chemical factors; it should be specific to a tissue; but most of all, it should be a fast, simple, accurate, reproducible and an economical method

[25]

. If Hanson and co-workers were the first team to introduce miRNA to body fluids identification in forensic field, soon enough other authors directed their work toward miRNA profiling. Figure 1 displays an overview of all miRNA that different authors considered as possible body-fluid biomarkers.

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56 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences In this figure it is emphasized the fact that almost all the studies performed had different outcomes and only few miRNAs are confirmed by two or more reports. Studies performed by Hanson, Zubakov, Wang et al. seem to point that miR-16 might be venous blood-specific, and consequently, a potential biomarker for blood stains

[16, 17, 20]

. Confirmed by three different studies, miR-205 was recognized as a good biomarker for saliva but Wang and colleagues concluded otherwise, sustaining that miR-205 may be epithelium specific and not saliva specific

[20]

. As a common act of vandalism, the urinating act can be a source of trace DNA but also particularly good for drug screening, especially drugs of abuse

[26, 27]

. Therefore, it is interesting to underline that no miRNA was detected as potential biomarker for urine, which is an important human fluid in forensics

[18]

. The lack of homogeneity and the non-reproducibility of results from different groups can be the outcome of dissimilar approaches to reach the one and same goal, highlighting the necessity of standardization for miRNA biomarker assessment.

Figure 1 - overview of all miRNA considered as possible body fluids biomarkers by [a] Hanson et al. [16]; [b]

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2.1 – Samples management and storing

The selection of adequate sample, sample processing and RNA extraction are crucial steps for miRNA profiling. The inclusion of degraded RNA in an experiment may result in the inability to detect and quantify specifics miRNA and enable the possibility to get reliable results [28]. However, it is now possible to extract high-quality miRNA from fresh tissues, cell lines, plasma, and serum among other body fluids to guarantee the quality and quantity of the sample, being both of them parameters that have significant impact on the result [18, 29].

Collecting samples is a very important stage to the entire profiling process. When collecting a sample, it might get contaminated with others than the pretended evidence – as ground soil, microorganisms, etc. It is common sense to believe that inadequate handling of the evidence can prevent or confuse the interpretation of the results. Another issue that needs to be raised is the effect of time exposure of the evidence to external factors until its collection and posterior extraction; we also have to consider the post-mortem interval. To our knowledge, there are no published studies that considered their potential effect on miRNAs profiling, nonetheless, we thought important to highlight those interrogations.

Nowadays, dried serum spots are commonly used for body fluid storage, namely blood. Can miRNAs be recovered in good conditions from dried samples? Patnaik et al. published an essay considering miRNA preservation in dried serum blots and they concluded that miRNA preservation was indeed reliable for a posterior profiling. However, they noticed that incomplete drying of blots before storing was prejudicial for its preservation [30].

Acknowledging that criminal investigation can take several years to resolve, can prior extracted miRNA remain in good conditions after mid-term / long-term storage? A very few studies worked to highlight this question with forensic relevant body fluids, though some work was made for human serum [31]. According to the study, four miRNAs were considered to evaluate the effect of storage in human serum at –80ºC (with and without thaw), –20ºC (with and without thaw) and at room temperature. Interestingly, mir-451 was one of the four miRNA considered and, as showed in figure1, it is also one regarded in venous blood. The authors concluded that no significant difference in miRNA levels was verified between –80ºC and –20ºC in short-term storage (10 days). However, when stored at roomed temperature, miRNA levels were drastically decreased, yet still detectable. Moreover, repeated freeze and thaw cycle also

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58 miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences significantly decrease miRNA levels compared with continuous storing, thought that result seems to be inconsistent with other authors research work results [32-34]. For mid-term storage (<20 month) no major differences on miRNA levels were observed between –80ºC and –20ºC, nonetheless some individual miRNA were seriously affected by those conditions. Finally, they explored the effect caused by long-term storage at –20ºC and observed a slightly decrease within the range of 2–4 years; after 6 years of storage a significant decrease of miRNA levels was perceived that only accentuates in the course of time [31]. In summary, miRNA seems to be almost unaffected by freezing temperatures (–20ºC) for at least 2–4 years in human serum, however it is indeed required a similar research work for others human body fluids as they may not respond the same way to such low temperature storage.

2.2 – MiRNA profiling – methodologies

MiRNAs proprieties bring a challenging game to its profiling, with more or less than 22 nucleotides in length (the size of a traditional primer) the traditional primer binding is impossible. In order to sidetrack the problem, the elaboration of a smaller primer is required, demanding a lower melting temperature and ultimately affecting the efficiency of the PCR. Furthermore, the short length and discrepancy in GC contents leads to a wide variance of melting temperatures resulting in another challenge to its reliable profiling.

Nowadays, a large cluster of methodologies has the capacity to be used for miRNA profiling. Methodologies like Northern Blotting using locked nucleic acid, RNase protection essays and in situ hybridization methodologies are known for miRNAs detection [35-37] . However, we decided to highlight the three major methods that are being currently used: microarray (hybridization-based method), qRT-PCR (quantitative reverse transcription PCR) and RNA sequencing. Interestingly, the groups taken in considerations for their work in miRNA profiling for body fluids identification used different combinations of methods. Could those combinations be responsible for the heterogeneity of results showed in figure 1? This hypothesis was suggested by Zubakov et al. when they tried to replicate the results obtained by Hanson and colleagues – for vaginal secretions, menstrual blood and saliva – and failed [17].

The concept of DNA microarray technology resides on the ability of a single strand of DNA (probe) to bind to a complementary strand of DNA (identified as target). The targets miRNA are initially reversed into cDNA and

miRNA as biomarker for body-fluids identification. Its role in Forensic Sciences

Sara dos Santos Teixeira da Silva

miRNA as biomarker for body-fluids identification

Its role in Forensic Sciences

Acknowledgment

Table of Contents

Abstract

Resumo

1 - Introduction

1.1 - Analytical mind

1.2 - Forensic Sciences

1.3 - Evolution of Forensic Science

1.4 - Forensic Genetics

1.4.1 - Study Case

1.4.2 - Genetic Background

1.5 - Importance of body fluids identification

1.5.1 - Blood Identification

1.5.2 - Semen Identification

1.5.3 - Saliva Identification

1.5.4 - Urine Identification

1.6 - Genetic approach – a broad confirmatory test?

1.6.1 - Potential of RNA

2 - Aims

3

Material

and

3 - Material and methods

Chapter 1 – Systematic Review:

“Forensic miRNA: Potential biomarker for body fluids?”

Chapter 2 – miRNA profiling:

“Circulating miRNA signatures for blood and urine identification”.

.

4 - Chapter 1

[

5]

[

11, 12]

[13]

[13]

[14]

[15]

[16]

[16-21]

1, 2, 4]

[22, 23]

[24]

[25]

[25]

[16, 17, 20]

[20]

[26, 27]

[18]

2.1 – Samples management and storing